JP7440724B2 - Antibodies that bind to CTLA4 and uses thereof - Google Patents

Description

Related Applications and Incorporation This application claims priority to U.S. Provisional Patent Application No. 63/008,931, filed April 13, 2020.

The above-mentioned application and all documents cited therein or during the prosecution thereof (the "Application Cited Documents") and all documents cited or referred to herein (all documents cited herein ("Application Cited Documents") (including, but not limited to, publications, patents, and published patent applications) (“Documents Cited herein”), and all documents cited or referenced in the documents cited herein, are incorporated herein by reference. Incorporated herein by reference, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any product described in the book or in any document incorporated by reference; It can be used to implement the present invention. More particularly, all referenced documents are incorporated by reference to the same extent as each individual document is specifically and individually indicated to be incorporated by reference. Any Genbank sequences described in this disclosure are incorporated by reference, along with the Genbank sequences as of the earliest effective filing date of this disclosure.

The present disclosure generally relates to isolated monoclonal antibodies, particularly murine, chimeric or humanized monoclonal antibodies, or antigen-binding portions thereof, that specifically bind human CTLA4 with high affinity and functionality. Nucleic acid molecules encoding the antibodies or antigen-binding portions thereof, expression vectors, host cells, and methods for expressing the antibodies or antigen-binding portions thereof are also provided. The present disclosure further provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses, and pharmaceutical compositions comprising antibodies or antigen-binding portions thereof, as well as anti-CTLA4 antibodies or antigen-binding portions thereof of the present disclosure. Provides a method for diagnosis or treatment using.

Immune checkpoints regulate the immune system and prevent it from attacking cells indiscriminately. Among the immune checkpoints, cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed death 1 (PD-1) are two important checkpoints that provide inhibitory signals during immune responses. For example, it has been reported that CTLA4 may arrest autoreactive T cells at an early stage of native T cell activation, whereas PD-1 regulates activated T cells at a later stage. I know that. These two inhibitory immune checkpoint pathways are also manipulated by tumor cells to evade immune system attack (Buchbinder EI, and Desai A. (2016) Am J Clin Oncol. 39(1):98 -106).

CTLA4 is a CD28 homolog and is primarily located in the intracellular compartment of resting naive T cells. It binds to CD80/CD86 with fairly high binding affinity, competing with CD28 on CD80/CD86 (Chambers CA et al., (2001) Annu Rev Immunol. 19:565-594). The CD28-CD80/CD86 interaction is important and sufficient for T-cell activation following binding of the TCR to major histocompatibility complex (MHC)-presented antigens on the surface of antigen-presenting cells (APCs). A high level of CD28-CD80/CD86 binding results in T cell proliferation, survival and differentiation (Buchbinder EI, and Desai A. (2016) Am J Clin Oncol. 39(1):98-106). CTLA4 translocates to the cell surface in the presence of stimulatory signals from TCR-TCR and CD28-CD80/86 interactions (Linsley PS et al., (1996) Immunity. 4:535-543) and CD80/ CTLA4 binding to CD86 generates either no stimulatory signal or even an inhibitory signal that counteracts the stimulatory signal from TCR-TCR and CD28-CD80/86 binding (Chambers CA et al., ( 2001) Annu Rev Immunol.19:565-594; Egen JG et al., (2002) Nat Immunol.3:611-618; Parry RV et al., (2005) Mol Cell Biol.25:9543-955 3; Fallarino F et al., (1998) J Exp Med. 188:205-210; Masterer EL et al., (2000) 164:5319-5327). Therefore, the relative amounts of CD28-CD80/86 binding and CTLA4-CD80/86 binding determine whether T cells undergo activation or anergy (Krummel MF, and Allison JP. (1995) J Exp Med. 182:459-465). CTLA4 can also lead to tryptophan catabolism and T cell proliferation inhibition by causing reverse signaling through CD80/CD86 and inducing indoleamine-2,3-dioxygenase (Boasso A et al., (2003). 2005) Blood 105:1574-1581).

CTLA-4 is also expressed on non-T cells, either normal or neoplastic cells (Laurent S et al., (2010) Hum Immunol 71:934-941; Contardi E et al., (2005) Int J Cancer 117:538-550). Persistent CTLA-4 expression in neoplastic cells contributes to hematology and solid tumor progression (Pistillo MP et al., (2003) Blood 101:202-209; Kosmaczewska A et al., (2005) Leukemia 19:301-304), CTLA4 pathway blockade has been found to be effective in reducing tumor growth (Leach DR et al., (1996) 271:1734-1736; Hirano F et al., (1996) 271:1734-1736). 2005) Cancer Res. 65:1089-1096). The anti-CTLA4 antibody ipilimumab (YERVOY®) is approved to treat melanoma, colorectal cancer, hepatocellular carcinoma, malignant pleural mesothelioma, non-small cell lung cancer, and renal cell carcinoma. Another anti-CTLA4 antibody, tremelimumab, is in clinical trials, for example, for the treatment of mesothelioma, melanoma, and colorectal cancer. CTLA-4 antibodies have also been used in combination with anti-PD-1 antibodies and/or other anti-tumor agents. For example, the anti-CTLA-4 antibody AGEN1884 is also an anti-PD-4 antibody used to treat cervical cancer, angiosarcoma, muscle-invasive bladder cancer, and soft tissue sarcomas (including synovial sarcomas, schwannomas, and phyllodes tumors). 1 antibody (National Cancer Institute).

Studies have further shown that CTLA-4 is upregulated in chronic infections, such as those caused by human immunodeficiency virus (HIV), and anti-CTLA-4 therapy alone or in combination with anti-PD-1 is not recommended in clinical trials. (Thomas A Rasmussen et al., (2021) Clinical Infectious Diseases ciaa1530; Colston E et al., (2018) PLoS One 13(6):e 0198158). Ipilimumab and nivolumab are also in Phase II trials for the treatment of Epstein-Barr virus (HHV-4) infection. Additionally, the effects of ipilimumab on graft-versus-host disease (GVHD), peripheral nerve injury, and neurofibromatosis type I (von Recklinghausen disease) are being explored in preclinical studies.

Continuing efforts are attempting to find more anti-CTLA4 binding moieties that are more potent or have more desirable properties.

Citation or identification of any document in this invention is not an admission that such document is available as prior art to this disclosure.

The present disclosure provides that CTLA4 binds to CTLA4 (e.g., human CTLA4 and monkey CTLA4) and has similar, if not higher, binding affinity for CTLA4, compared to prior art anti-CTLA4 antibodies such as ipilimumab, CTLA4- An isolated monoclonal antibody, such as a murine, human, chimeric, or humanized monoclonal antibody or its Provides an antigen binding moiety.

The antibodies or antigen-binding portions of the present disclosure can be used in a variety of applications, including the detection of CTLA4 protein and the treatment and prevention of CTLA4-related diseases such as cancer and infectious diseases.

Accordingly, in one aspect, the present disclosure provides a heavy chain variable region that may include i) a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, wherein the VH CDR1 region, the VH CDR2 region, and the VH CDR3 region are comprised of (1) Each of SEQ ID NO: 1, 2 and 3; (2) Each of SEQ ID NO: 7, 8 and 9; (3) Each of SEQ ID NO: 1, 2 and 13; (4) Each of SEQ ID NO: 16, 17 and 18; ( 5) Each of SEQ ID NO: 22, 23 and 24; (6) Each of SEQ ID NO: 28, 29 and 30; (7) Each of SEQ ID NO: 22, 34 and 24; (8) Each of SEQ ID NO: 36, 37 and 38 (9) each of SEQ ID NO: 42, 43 and 44; or (10) at least 85%, 86%, 87%, 88%, 89%, 90%, 91 for each of SEQ ID NO: 48, 49 and 50. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity; and/or ii) the VL A light chain variable region that may include a CDR1 region, a VL CDR2 region, and a VL CDR3 region, wherein the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region are (1) each of SEQ ID NOs: 4, 5, and 6; (2) Each of SEQ ID NO: 10, 11 and 12; (3) Each of SEQ ID NO: 14, 5 and 15; (4) Each of SEQ ID NO: 19, 20 and 21; (5) Each of SEQ ID NO: 25, 26 and 27; ( 6) Each of SEQ ID NO: 31, 32 and 33; (7) Each of SEQ ID NO: 35, 26 and 27; (8) Each of SEQ ID NO: 39, 40 and 41; (9) Each of SEQ ID NO: 45, 46 and 47 or (10) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% for each of SEQ ID NOs: 51, 52 and 53. An isolated monoclonal antibody (e.g., murine, chimeric, or humanized antibodies) or antigen-binding portions thereof.

The antibodies or antigen-binding portions thereof of the present disclosure include a heavy chain variable region that may include a VH CDR1 region, a VH CDR2 region, and a VH CDR3 region, and a light chain variable region that may include a VL CDR1 region, a VL CDR2 region, and a VL CDR3 region. (1) each of SEQ ID NOs: 1, 2, 3, 4, 5, and 6; Each of SEQ ID NO: 7, 8, 9, 10, 11 and 12; (3) Each of SEQ ID NO: 1, 2, 13, 14, 5 and 15; (4) SEQ ID NO: 16, 17, 18, 19, 20 and (5) Each of SEQ ID NO: 22, 23, 24, 25, 26 and 27; (6) Each of SEQ ID NO: 28, 29, 30, 31, 32 and 33; (7) SEQ ID NO: 22, 34 , 24, 35, 26 and 27; (8) each of SEQ ID NO: 36, 37, 38, 39, 40 and 41; (9) each of SEQ ID NO: 42, 43, 44, 45, 46 and 47; or (10) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 for each of SEQ ID NOs: 48, 49, 50, 51, 52, and 53; %, 95%, 96%, 97%, 98%, 99% or 100% identity, the antibody or antigen-binding fragment thereof binds to CTLA4.

The heavy chain variable regions of the antibodies or antigen-binding portions thereof of the present disclosure include 54,55 (X1=A, X2=V; A), 58, 59 (X1=P, X2=A, X3=D; X1=L, X2=A, X3=N; X1=L, X2=A, X3=D; X1=L, X2=S , %, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, the antibody or antigen-binding portion thereof binds to CTLA4. The amino acid sequence of SEQ ID NO: 54 may be encoded by the nucleotide sequence of SEQ ID NO: 80 or 81. The amino acid sequence of SEQ ID NO: 58 may be encoded by the nucleotide sequence of SEQ ID NO: 86 or 87. The amino acid sequences of SEQ ID NO: 55 (X1=G, X2=A) and 59 (X1=P, X2=A, X3=D) can be encoded by the nucleotide sequences of SEQ ID NO: 82 and 88, respectively.

The light chain variable region of the antibody or antigen-binding portion thereof of the present disclosure is SEQ ID NO: 56, 57 (X1=S, X2=M, X3=R, X4=Y; X1=S, X2=V, X3=T, X4=F; X1=V, X2=V, X3=T, X4=F), 60, 61 (X1=T, X2=V, =V, X2=P, %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, the antibody or antigen-binding portion thereof binds to CTLA4. . The amino acid sequence of SEQ ID NO: 56 may be encoded by the nucleotide sequence of SEQ ID NO: 83 or 84. The amino acid sequence of SEQ ID NO: 60 may be encoded by the nucleotide sequence of SEQ ID NO: 89 or 90. The amino acid sequences of SEQ ID NO: 57 (X1=S, X2=V, X3=T, X4=F) and 61 (X1=V, X2=P, X3=Y) are the nucleotide sequences of SEQ ID NO: 85 and 91, respectively. can be coded by

The antibodies or antigen-binding portions thereof of the present disclosure include (1) SEQ ID NOs: 54 and 56, respectively; (2) SEQ ID NOs: 55 (X1=A, X2=V) and 57 (X1=S, X2=M, X3= R, X4=Y); (3) each of SEQ ID NO: 55 (X1=A, X2=A) and 57 (X1=S, X2=M, X3=R, X4=Y); (4) Sequence Numbers 55 (X1=G, X2=V) and 57 (X1=S, X2=M, X3=R, X4=Y), respectively; (5) Sequence number 55 (X1=G, X2=A) and 57 (X1=S, X2=M, X3=R, X4=Y); (6) Sequence number 55 (X1=A, X2=V) and 57 (X1=S, X2=V, (7) each of SEQ ID NO: 55 (X1 = A, X2 = A) and 57 (X1 = S, X2 = V, X3 = T, X4 = F); (8) SEQ ID NO: 55 (X1=G, X2=V) and 57 (X1=S, X2=V, X3=T, = S, X2 = V, X3 = T, X4 = F); (10) Sequence number 55 (X1 = A, X2 = V) and 57 (X1 = V, X2 = V, X3 = T, X4 = (11) Each of SEQ ID NO: 55 (X1=A, X2=A) and 57 (X1=V, X2=V, X3=T, X4=F); (12) SEQ ID NO: 55 (X1 = G, X2 = V) and 57 (X1 = V, X2 = V, X3 = T, X4 = F), respectively; , X2=V, X3=T, X4=F); (14) SEQ ID NO: 58 and 60, respectively; (15) SEQ ID NO: 59 (X1=P, =T, X2=V, X3=F); (16) each of SEQ ID NO: 59 (X1=L, X2=A, X3=N) and 61 (X1=T, X2=V, X3=F) (17) Each of SEQ ID NO: 59 (X1=L, X2=A, X3=D) and 61 (X1=T, X2=V, X3=F); (18) SEQ ID NO: 59 (X1=L, X2 =S, X3=N) and 61 (X1=T, X2=V, X3=F), respectively; (19) SEQ ID NO: 59 (X1=P, , X2=P, X3=F); (20) Sequence number 59 (X1=L, X2=A, X3=N) and 61 (X1=V, X2=P, X3=F); 21) SEQ ID NO: 59 (X1=L, X2=A, X3=D) and 61 (X1=V, X2=P, X3=F), respectively; (22) SEQ ID NO: 59 (X1=L, X2=S , X3=N) and 61 (X1=V, X2=P, X3=F); (23) SEQ ID NO: 59 (X1=P, X2=A, = P, X3 = Y); (24) each of SEQ ID NO: 59 (X1 = L, X2 = A, X3 = N) and 61 (X1 = V, X2 = P, X3 = Y); (25) Each of SEQ ID NO: 59 (X1=L, X2=A, X3=D) and 61 (X1=V, X2=P, X3=Y); (26) SEQ ID NO: 59 (X1=L, X2=S, X3 =N) and 61 (X1=V, X2=P, (30) Each of SEQ ID NO: 68 and 69; (31) Each of SEQ ID NO: 70 and 71; (32) Each of SEQ ID NO: 72 and 73; (33) Each of SEQ ID NO: 74 and 75; or ( 34) at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 for each of SEQ ID NOs: 76 and 77; %, 98%, 99% or 100% identity.

An isolated monoclonal antibody or antigen-binding portion thereof of the present disclosure may include a heavy chain and a light chain linked by a disulfide bond, the heavy chain may include a heavy chain variable region and a heavy chain constant region, and the light chain may include a heavy chain variable region and a heavy chain constant region. may include a light chain variable region and a light chain constant region, wherein the C-terminus of the heavy chain variable region is linked to the N-terminus of the heavy chain constant region, and the C-terminus of the light chain variable region is linked to the N-terminus of the light chain constant region. , the heavy chain variable region and the light chain variable region can include the amino acid sequences described above, and the antibody or antigen-binding portion thereof binds to CTLA4. The heavy chain constant region can be an IgG1, IgG2 or IgG4 heavy chain constant region, eg, a human IgG4 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 78 and the like. Heavy chain constant regions, such as Fc fragments, can be engineered to have reduced or enhanced FcR binding affinity. The light chain constant region can be a kappa constant region, eg, a human kappa constant region having the amino acid sequence shown in SEQ ID NO: 79 and the like. The amino acid sequences of SEQ ID NOs: 78 and 79 can be encoded by the nucleotide sequences of SEQ ID NOs: 92 and 93, respectively.

Antibodies of the present disclosure in certain embodiments may comprise or consist of two heavy chains and two light chains, each heavy chain comprising the heavy chain constant region, heavy chain variable region or CDR sequences described above. and each light chain may include a light chain constant region, light chain variable region or CDR sequences described above, and the antibody binds to CTLA4. An antibody or antigen-binding portion thereof of the present disclosure can be a full-length antibody, such as an IgG1, IgG2, or IgG4 isotype. In other embodiments, the disclosed antibodies or antigen-binding portions thereof can be single chain variable fragment (scFv) antibodies or antibody fragments, such as Fab or F(ab') ₂ fragments.

The present disclosure can also include an antibody or antigen-binding portion thereof of the present disclosure linked to a second functional moiety (e.g., a second antibody) that has a different binding specificity than said antibody or antigen-binding portion thereof. Provides bispecific molecules. The present disclosure also provides immunoconjugates, such as antibody-drug conjugates, which may include an antibody of the present disclosure, or antigen-binding portion thereof, linked to a therapeutic agent, such as a cytotoxin. In another aspect, the antibodies of the present disclosure, or antigen-binding portions thereof, can be produced as part of a chimeric antigen receptor (CAR). Also provided are immune cells, such as T cells and NK cells, which can include antigen chimeric receptors. The antibodies or antigen-binding portions thereof of the present disclosure can also be encoded by or used in conjunction with oncolytic viruses.

Nucleic acid molecules encoding antibodies of the present disclosure or antigen-binding portions thereof, as well as expression vectors that may contain such nucleic acids and host cells that may contain such expression vectors, are also encompassed by this disclosure. Also provided are methods of preparing the anti-CTLA4 antibodies or antigen-binding portions thereof of the present disclosure using host cells. The method can include (i) expressing the antibody in the host cell and (ii) isolating the antibody from the host cell or cell culture thereof.

an antibody or antigen-binding portion thereof, immunoconjugate, bispecific molecule, oncolytic virus, CAR, CAR-T cell, nucleic acid molecule, expression vector or host cell of the present disclosure; and a pharmaceutically acceptable carrier. Also provided are compositions that can include. In certain embodiments, the pharmaceutical composition may further contain a therapeutic agent, such as an anti-cancer agent.

In yet another aspect, the present disclosure provides a method of modulating an immune response in a subject, comprising: administering a therapeutically effective amount of an antibody of the present disclosure or an antigen-binding portion thereof or an alternative antibody such that the immune response is modulated in the subject. The method comprises administering to a subject nucleic acid molecules capable of expressing them in the subject. Preferably, the antibodies or antigen-binding portions thereof of the present disclosure potentiate, stimulate, or increase an immune response in a subject.

In yet another aspect, the present disclosure provides a method of inhibiting tumor growth in a subject in need thereof, comprising a therapeutically effective amount of an antibody or antigen-binding portion thereof of the present disclosure or alternatively the ability to express the same in a subject. A method comprising administering a nucleic acid molecule to a subject is provided. In some embodiments, the method comprises administering an oncolytic virus encoding or carrying an antibody, a bispecific molecule, immunoconjugate, CAR-T cell, or antibody of the present disclosure. The tumor may be a solid or hematological tumor. In certain embodiments, the tumor is, but is not limited to, melanoma, colorectal cancer, hepatocellular carcinoma, pleural mesothelioma, lung cancer (e.g., non-small cell lung cancer), renal cell carcinoma, cervical cancer. , angiosarcoma, malignant pleural mesothelioma, metastatic transitional (urothelial) urinary tract cancer, ureteral cancer, urethral cancer, urinary tract cancer, head and neck cancer, squamous cell carcinoma, transitional cell carcinoma (urothelial cell carcinoma) , esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, esophagogastric junction adenocarcinoma, anal cancer, bile duct cancer (cholangiocarcinoma), dysgerminoma, endometrial cancer, fallopian tube cancer, germ cell tumor , myelodysplastic syndromes, neuroblastoma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, peritoneal cancer, prostate cancer, salivary gland cancer, sarcoma, triple negative breast cancer (TNBC), and solid tumors including muscle-invasive bladder cancer . In some embodiments, an anti-VISTA antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-STAT3 antibody and /or at least one additional anti-cancer antibody such as an anti-ROR1 antibody can be administered. In yet another embodiment, the antibodies of the present disclosure or antigen-binding portions thereof are cytokines (e.g., IL-2, IL-21, GM-CSF and/or IL-4) or costimulatory antibodies (e.g., anti-CD137 and or anti-GITR antibody). In another embodiment, the antibodies of the present disclosure or antigen-binding portions thereof are administered with a chemotherapeutic agent that can be a cytotoxic agent such as epirubicin, oxaliplatin, and/or 5-fluorouracil (5-FU). The antibodies or antigen-binding portions thereof of the present disclosure can be, for example, murine, human, chimeric, or humanized.

In another aspect, the present disclosure provides a method of treating or alleviating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present disclosure. Infectious diseases can be diseases caused by viral, bacterial, fungal or mycoplasma infections. In certain embodiments, the infectious disease is caused by chronic HIV infection or HHV-4 infection. In certain embodiments, the subject may further be administered at least one anti-infective agent, such as an anti-viral, anti-bacterial, anti-fungal or anti-mycoplasmal agent.

Other features and advantages of the present disclosure will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Therefore, it is not the purpose of the present invention to encompass within the present invention any already known products, methods of making the products, or methods of using the products, and the applicant therefore reserves the right to Disclosure of any previously known products, processes, or methods is disclosed herein. This invention relates to any product, process, or product that does not meet the written description and enablement requirements of the USPTO (U.S. Pat. It is further noted that it is not intended to encompass within the scope of the present invention the methods of making or the methods of use of the product, and therefore the applicant reserves the right to include any already described product, the product Disclosed herein are methods of making or using the products. In the practice of the invention, it may be advantageous to comply with Article 53(c) EPC and Regulation 28(b) and (c) EPC. All rights to specifically disclaim any embodiments that are the subject matter of any registered patent of Applicant in the family of this application or any other family or any prior application of any third party are expressly reserved. be done. Nothing stated herein should be construed as a warranty.

In this disclosure, particularly in the claims and/or paragraphs, terms such as "comprises," "comprised," "comprising," and the like may have the meaning according to U.S. patent law. for example, they can mean "includes", "included", "including", etc.; and "consisting essentially of" and "from" Terms such as "consists essentially of" have the meaning according to U.S. patent law, e.g., they allow for elements not expressly identified but found in the prior art or that form the basis of the invention. It is noted that elements that affect specific or novel features are excluded.

BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description, given by way of example and not intended to limit the invention to only the particular embodiments described, may be best understood in conjunction with the accompanying drawings.

Figure 3 shows the binding capacity of mouse antibodies D1H4, D1A7 and D1B6 to human CTLA4 in a capture ELISA. Figure 3 shows the binding capacity of mouse antibodies C1G4, D1H3, D1B8, D1D5, D2A4, C1D1 and D1G6 against human CTLA4 in a capture ELISA. Figure 3 shows the ability of mouse antibodies D2A4, C1D1, C1G4, D1B6 and D1D5 to block benchmark binding to human CTLA4 in a competitive ELISA. Figure 3 shows the ability of mouse antibodies D1H3, D1A7, D1G6, D1H4 and D1B8 to block benchmark binding to human CTLA4 in a competitive ELISA. Figure 3 shows the ability of murine antibodies D1H4, D1A7, and D1B6 to block CTLA4 binding to cell surface CD80/CD86 in a cell-based blocking FACS assay. Figure 3 shows the ability of murine antibodies C1G4, D1H3, D1B8, D1D5, D2A4, C1D1 and D1G6 to block CTLA4 binding to cell surface CD80/CD86 in a cell-based blocking FACS assay. We show that murine antibodies D2A4, C1D1, C1G4, D1B6, D1D5, D1H3, D1H4, D1G6, D1B8 and D1A7 blocked CTLA4-CD80 binding and induced IL-2 release in a cell-based functional assay. Figure 2 shows the binding ability of chimeric antibody C1G4 to human CTLA4 in capture ELISA. Figure 3 shows the binding ability of chimeric antibody D1B6 to human CTLA4 in a capture ELISA. Figure 2 shows the binding ability of chimeric antibody C1D1 to human CTLA4 in capture ELISA. Figure 2 shows the binding ability of chimeric antibody D1D5 to human CTLA4 in a capture ELISA. Figure 2 shows the binding ability of chimeric antibody D1B8 to human CTLA4 in a capture ELISA. Figure 3 shows the ability of chimeric antibodies C1G4, D1B6 and C1D1 to block CTLA4 binding to cell surface CD80/CD86 in a cell-based blocking FACS assay. Figure 3 shows the ability of chimeric antibodies D1D5 and D1B8 to block CTLA4 binding to cell surface CD80/CD86 in a cell-based blocking FACS assay. Figure 3 shows the binding ability of humanized antibody huC1D1-V8 to human CTLA4 in a capture ELISA. Figure 3 shows the binding ability of humanized antibody huD1D5-V9 to human CTLA4 in a capture ELISA. Benchmark in competitive ELISA - shows the ability of humanized antibody huC1D1-V8 to block human CTLA4 binding. Benchmark in competitive ELISA - shows the ability of humanized antibody huD1D5-V9 to block human CTLA4 binding. Figure 3 shows the ability of humanized antibody huC1D1-V8 to block CTLA4 binding to cell surface CD80/CD86 in a cell-based blocking FACS assay. Figure 3 shows the ability of humanized antibody huD1D5-V9 to block CTLA4 binding to cell surface CD80/CD86 in a cell-based blocking FACS assay. We show that humanized antibodies huC1D1-V8 and huD1D5-V9 blocked CTLA4-CD80 binding and induced IL-2 release in a cell-based functional assay. The results of protein thermal shift assay of humanized antibody huC1D1-V8 are shown. The results of protein thermal shift assay of humanized antibody huD1D5-V9 are shown.

To ensure that this disclosure can be more easily understood, some terms are first defined. Further definitions are provided throughout the detailed description.

The term "CTLA4" refers to cytotoxic T lymphocyte-associated antigen 4. The term "CTLA4" includes variants, isoforms, homologs, orthologs and paralogs. For example, antibodies specific for human CTLA4 protein may in some cases cross-react with CTLA4 protein from non-human species, such as monkeys. In other embodiments, antibodies specific for human CTLA4 protein may be completely specific for human CTLA4 protein and may exhibit no other species or other types of cross-reactivity, or all It may cross-react with CTLA4 from certain but not other species.

The term "human CTLA4" refers to a CTLA4 protein having an amino acid sequence derived from a human, such as the amino acid sequence of human CTLA4 having a Genbank accession number of NP_005205. The terms "monkey or rhesus CTLA4" and "mouse CTLA4" refer to monkey and mouse CTLA4 sequences, respectively, eg, those having amino acid sequences having Genbank accession numbers NP_001038204.1 and NP_033973.2, respectively.

The term "immune response" refers to selective damage to invading pathogens, pathogen-infected cells or tissues, cancerous cells or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues, their destruction or refers to the action of soluble macromolecules (including antibodies, cytokines and complement) produced by, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes and other cells or the liver, resulting in their elimination from the body do.

The term "antibody" as used herein refers to an immunoglobulin molecule that recognizes and specifically binds to a target, such as CTLA4, through at least one antigen-binding site; The binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term includes intact polyclonal antibodies, intact monoclonal antibodies, single chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs), light chain antibodies (LCAbs), multispecific antibodies. , bispecific antibodies, monospecific antibodies, monovalent antibodies, fusion proteins containing an antigen-binding site of an antibody, and any other protein containing an antigen-binding site as long as the antibody exhibits the desired biological activity. Modified immunoglobulin molecules (eg, dual variable domain immunoglobulin molecules) are included. Antibodies also include, but are not limited to, murine antibodies, chimeric antibodies, humanized antibodies and human antibodies. Antibodies are divided into five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM or their subclasses ( isotype) (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). Different classes of immunoglobulins have different well-known subunit structures and three-dimensional arrangements. Antibodies can be naked or conjugated to other molecules, including, but not limited to, toxins and radioisotopes. Unless explicitly specified otherwise, the term "antibody" as used herein includes the "antigen-binding portion" of an intact antibody. IgG is a glycoprotein that may contain two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain may be composed of a heavy chain variable region (abbreviated herein as _VH ) and a heavy chain constant region. The heavy chain constant region may be composed of three domains, C _H1 , C _H2 and C _H3 . Each light chain may be composed of a light chain variable region (abbreviated herein as _VL ) and a light chain constant region. The light chain constant region may be composed of one domain, _CL . The V _H and V _L regions can be further divided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with more conserved regions called framework regions (FR). Each V _H and V _L is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of heavy and light chains contain binding domains that interact with antigen. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q).

As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to an antibody that retains the ability to specifically bind an antigen (e.g., CTLA4 protein). Refers to the above fragment. It has been shown that the antigen binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of V _L , V _H , C _L and C _H1 domains; (ii) F(ab' ) ₂ fragment, a bivalent fragment that may contain two Fab fragments connected by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the V _H and C _H1 domains; (iv) the V _L of a single arm of the antibody. (v) a dAb fragment consisting of a V _H domain (Ward _et al., (1989) Nature 341:544-546); (vi) an isolated complementarity determining region (CDR); and (viii) a Nanobody, comprising a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they are combined into a single domain in which the V _L and V _H regions pair to form a monovalent molecule. Protein chains (known as single chain Fvs (scFvs); eg, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883) can be joined using recombinant methods, by means of synthetic linkers that allow for the production of nucleotides (see US 85:5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and these fragments are screened for utility in the same manner as intact antibodies.

"Isolated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., An isolated antibody is substantially free of antibodies that specifically bind to antigens other than CTLA4 protein). However, isolated antibodies that specifically bind human CTLA4 protein may have cross-reactivity to other antigens, such as CTLA4 proteins from other species. Furthermore, an isolated antibody may be substantially free of other cellular materials and/or chemicals.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of single molecule composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "murine antibody" as used herein is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from murine germline immunoglobulin sequences. Additionally, if the antibody includes a constant region, the constant region is also derived from mouse germline immunoglobulin sequences. The murine antibodies of the present disclosure may contain amino acid residues that are not encoded by murine germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). However, the term "murine antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto murine framework sequences. Not yet.

The term "chimeric antibody" refers to an antibody produced by combining genetic material derived from a non-human source with genetic material derived from a human. Or, more generally, a chimeric antibody is an antibody that has genetic material from a particular species together with genetic material from another species.

As used herein, the term "humanized antibody" is derived from a non-human species in which the protein sequence has been modified to increase its similarity to the antibody form naturally produced in humans. Refers to antibodies.

The term "isotype" refers to the antibody class (eg, IgM or IgG1) encoded by the heavy chain constant region genes.

The phrases "antigen-recognizing antibody" and "antigen-specific antibody" are used herein interchangeably with the term "antigen-specifically binding antibody."

As used herein, an antibody that "specifically binds human CTLA4" binds to a human CTLA4 protein (optionally one or more non-human derived CTLA4 proteins), but not to a non-CTLA4 protein. It is intended to refer to antibodies that do not substantially bind. Preferably, the antibody is "high affinity", ie, 5.0×10 ⁻⁸ M or less, more preferably 1.0×10 ⁻⁸ M or less, more preferably 7.0×10 ⁻⁹ M Binds to human CTLA4 protein with the following _KD .

As used herein, the term "does not substantially bind" to a protein or cell refers to not binding or not binding with high affinity to the protein or cell, i.e., 1.0 x 10 ^-6 M Above, more preferably 1.0×10 ⁻⁵ M or more, more preferably 1.0×10 ⁻⁴ M or more, more preferably 1.0×10 ⁻³ M or more, even more preferably 1 It means binding to proteins or cells with a K _D of .0×10 ⁻² M or more.

The term "high affinity" for an IgG antibody means 1.0×10 ⁻⁶ M or less, more preferably 5.0×10 ⁻⁸ M or less, even more preferably 1.0× Refers to an antibody having a K _D of 10 ⁻⁸ M or less, even more preferably 7.0×10 ⁻⁹ M or less, even more preferably 1.0×10 ⁻⁹ M or less. However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding to an IgM isotype refers to an antibody with a K _D of 10 ⁻⁶ M or less, more preferably 10 ⁻⁷ M or less, even more preferably 10 ⁻⁸ M or less.

While the term "K _assoc " or "K _a " as used herein is intended to refer to the binding rate of a particular antibody-antigen interaction, as used herein The term “K _dis ” or “K _d ” is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term “K _D ” as used herein is intended to refer to the dissociation constant, which is obtained from the ratio of K _d to _Ka (i.e., K _d /K _a ) and is Expressed as concentration (M). K _D values for antibodies can be determined using methods well established in the art. A preferred method for determining the K _D of an antibody uses surface plasmon resonance, preferably by using a biosensor system such as the Biacore™ system.

The term " _EC50 ," also known as half-maximal effective concentration, refers to the concentration of antibody that induces a response intermediate between baseline and maximal after a specified exposure time.

The term " _IC50 ," also known as half-inhibitory concentration, refers to the concentration of an antibody that inhibits a particular biological or biochemical function by 50% compared to the absence of the antibody.

The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, including mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, but Mammals such as non-human primates, sheep, dogs, cats, cows and horses are preferred.

The term "therapeutically effective amount" refers to an amount of an antibody of the present disclosure sufficient to prevent or ameliorate symptoms associated with a disease or condition (such as cancer) and/or reduce the severity of the disease or condition. means. A therapeutically effective amount will be understood with respect to the condition being treated, and an actual effective amount will be readily understood by one of ordinary skill in the art.

Various aspects of the disclosure are described in further detail in the following subsections.

The antibodies or antigen-binding portions thereof of the present disclosure specifically bind human CTLA4 with comparable, if not better, binding affinity/ability compared to previously described anti-CTLA4 antibodies such as ipilimumab.

The antibodies or antigen-binding portions thereof of the present disclosure block CTLA4 binding to CD80/CD86 with equal or higher activity compared to previously described anti-CTLA4 antibodies such as ipilimumab. Antibodies or antigen-binding portions thereof of the present disclosure promote T cell responses that are inhibited by CTLA4-CD80/CD86 binding.

The antibodies or antigen-binding portions thereof of the present disclosure are murine, chimeric or humanized.

The antibodies or antigen-binding portions thereof of the present disclosure are structurally and chemically characterized as described below and in the Examples below. Amino acid sequence ID numbers for the heavy/light chain variable regions of antibodies are summarized in Table 1 below, and several antibodies share the same V _H or V _L. The heavy chain constant region of the antibody can be, for example, a human IgG4 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 78, and the light chain constant region of the antibody can be, for example, a human IgG4 heavy chain constant region having the amino acid sequence shown in SEQ ID NO: 79. It can be a constant region. These antibodies may also contain a mouse IgG4 heavy chain constant region and a mouse kappa constant region.

The heavy chain variable region CDRs and light chain variable region CDRs in Table 1 are defined by the Kabat numbering system. However, as is well known in the art, CDR regions may also be determined by other systems such as Chothia, IMGT, AbM, and Contact numbering systems/methods based on heavy chain/light chain variable region sequences. obtain.

The V _H and V _L sequences (or CDR sequences) of other anti-CTLA4 antibodies that bind to human CTLA4 are "mixed and matched" with the V _H and V _L sequences (or CDR sequences) of the anti-CTLA4 antibodies of the present disclosure. I can do it. Preferably, when the V _H and V _L chains (or CDRs within such chains) are mixed and matched, the V _H sequences from a particular V _H /V _L pairing are Replaced with _H array. Similarly, preferably the V _L sequence from a particular V _H /V _L pairing is replaced with a structurally similar V _L sequence.

Thus, in one embodiment, the antibodies of the present disclosure, or antigen-binding portions thereof, are
(a) a heavy chain variable region comprising the amino acid sequence listed above in Table 1; and (b) a light chain variable region comprising the amino acid sequence listed above in Table 1, or of another anti-CTLA4 antibody. V _L , where the antibody specifically binds to human CTLA4.

In another embodiment, the antibodies of the present disclosure, or antigen-binding portions thereof, are
(a) the CDR1, CDR2, and CDR3 regions of the heavy chain variable regions listed above in Table 1; and (b) the CDR1, CDR2, and CDR3 regions of the light chain variable regions listed above in Table 1. or the CDRs of another anti-CTLA4 antibody, where the antibody specifically binds human CTLA4.

In yet another embodiment, the antibody, or antigen-binding portion thereof, comprises the heavy chain of an anti-CTLA4 antibody in combination with the CDRs of another antibody that binds to human CTLA4, such as CDR1 and/or CDR3 from the heavy chain variable region. variable CDR2 regions, and/or CDR1, CDR2, and/or CDR3 from the light chain variable regions of different anti-CTLA4 antibodies.

Furthermore, independent of the CDR1 and/or CDR2 domains, the CDR3 domain alone can determine the binding specificity of an antibody to a cognate antigen, and as expected, multiple antibodies bind to a common CDR3 sequence. It is well known in the art that the same binding specificity can be produced based on the same binding specificity. For example, Klimka et al. , British J. of Cancer 83(2):252-260 (2000); Beiboer et al. , J. Mol. Biol. 296:833-849 (2000); Rader et al. , Proc. Natl. Acad. Sci. U. S. A. 95:8910-8915 (1998); Barbas et al. , J. Am. Chem. Soc. 116:2161-2162 (1994); Barbas et al. , Proc. Natl. Acad. Sci. U. S. A. 92:2529-2533 (1995); Ditzel et al. , J. Immunol. 157:739-749 (1996); Berezov et al. , BIA journal 8: Scientific Review 8 (2001); Igarashi et al. , J. Biochem (Tokyo) 117:452-7 (1995); Bourgeois et al. , J. Virol 72:807-10 (1998); Levi et al. , Proc. Natl. Acad. Sci. U. S. A. 90:4374-8 (1993); Polymenis and Stoller, J. Immunol. 152:5218-5329 (1994) and Xu and Davis, Immunity 13:37-45 (2000). Also, US Pat. No. 6,951,646; US Pat. No. 6,914,128; US Pat. No. 6,090,382; US Pat. , 156,313; 6,827,925; 5,833,943; 5,762,905 and 5,760,185 Please refer to the book. Each of these references is incorporated herein by reference in its entirety.

Thus, in another embodiment, an antibody of the present disclosure comprises CDR2 of the heavy chain variable region of an anti-CTLA4 antibody, and at least CDR3 of the heavy chain and/or light chain variable region of an anti-CTLA4 antibody, or of another anti-CTLA4 antibody. CDR3 of the heavy chain and/or light chain variable region, where the antibody is capable of specifically binding human CTLA4. These antibodies preferably (a) compete for binding with CTLA4; (b) retain functional properties; (c) bind to the same epitope; and/or (d) anti-CTLA4 antibodies of the present disclosure. has similar binding affinity. In yet another embodiment, the antibody may further comprise the CDR2 of the light chain variable region of an anti-CTLA4 antibody, or the CDR2 of the light chain variable region of another anti-CTLA4 antibody, wherein the antibody is specific for human CTLA4. It is possible to combine with In another embodiment, an antibody of the present disclosure may further comprise a CDR1 of a heavy chain and/or light chain variable region of an anti-CTLA4 antibody, or a CDR1 of a heavy chain and/or light chain variable region of another anti-CTLA4 antibody. , wherein the antibody is capable of specifically binding human CTLA4.

Conservative Modifications In another embodiment, the antibodies of the present disclosure have heavy and/or light chain variable region CDR1, CDR2, and CDR3 sequences that differ from those of the anti-CTLA4 antibodies of the present disclosure by one or more conservative modifications. Contains arrays. It is understood in the art that certain conservative sequence modifications can be made without eliminating antigen binding. For example, Brummell et al. , (1993) Biochem 32:1180-8; de Wildt et al. , (1997) Prot. Eng. 10:835-41; Komissarov et al. , (1997) J. Biol. Chem. 272:26864-26870; Hall et al. , (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conqui et al. , (1998) Int. Immunol. 10:341-6 and Beers et al. , (2000) Clin. Can. Res. 6:2835-43.

Thus, in one embodiment, the antibody comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and/or a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein:
(a) the heavy chain variable region CDR1 sequences include the sequences listed in Table 1 above, and/or conservative modifications thereof; and/or (b) the heavy chain variable region CDR2 sequences include the sequences listed in Table 1 above; (c) the heavy chain variable region CDR3 sequence comprises the sequences listed in Table 1 above, and/or conservative modifications thereof; (d) the light chain variable region CDR1, and/or CDR2, and/or CDR3 sequences include the sequences listed in Table 1 above; and/or conservative modifications thereof;
(e) The antibody specifically binds to human CTLA4.

Antibodies of the present disclosure have one or more of the following functional properties described above, such as high affinity binding to human CTLA4 and blocking activity of CTLA4-CD80/CD86 binding.

In various embodiments, the antibody can be, for example, murine, human, humanized or chimeric.

As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not substantially affect or alter the binding properties of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine). , cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of the antibodies of the present disclosure can be substituted with other amino acid residues of the same side chain family, and the modified antibodies can be used in the functional assays described herein. can be tested for retained functionality (i.e., the functionality described above) using

Antibodies of the present disclosure can be prepared using antibodies having one or more of the V _H /V _L sequences of the anti-CTLA4 antibodies of the present disclosure as a starting material to engineer modified antibodies. The antibody contains one or more residues within one or both variable regions (i.e., V _H and/or V _L ), e.g., within one or more CDR regions and/or within one or more framework regions. can be manipulated by modifying. Additionally or alternatively, antibodies can be engineered by modifying residues within the constant region, eg, to alter the effector functions of the antibody.

In certain embodiments, CDR grafting can be used to engineer variable regions of antibodies. Antibodies interact with target antigens primarily through amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs are more different between individual antibodies than the sequences outside the CDRs. Because CDR sequences are involved in most antibody-antigen interactions, specific It is possible to express recombinant antibodies that mimic the properties of natural antibodies (e.g., Riechmann et al., (1998) Nature 332:323-327; Jones et al., (1986) Nature 321:522- 525; Queen et al., (1989) Proc. Natl. Acad. U.S.A. 86:10029-10033; U.S. Pat. , 101; see also 5,585,089; 5,693,762 and 6,180,370).

Accordingly, another embodiment of the present disclosure provides a heavy chain variable region comprising a CDR1, CDR2, and CDR3 sequence comprising a sequence of the present disclosure, as described above, and/or a CDR1 comprising a sequence of the present disclosure, as described above. An isolated monoclonal antibody, or antigen-binding portion thereof, comprising a light chain variable region comprising CDR2 and CDR3 sequences. While these antibodies contain the V _H and V _L CDR sequences of the monoclonal antibodies of the present disclosure, they may contain different framework sequences.

Such framework sequences can be obtained from public DNA databases or published references containing germline antibody gene sequences. For example, the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk/vbase). , as well as Kabat et al. , (1991) (cited above); Tomlinson et al. , (1992) J. Mol. Biol. 227:776-798; and Cox et al. , (1994) Eur. J. Immunol. 24:827-836, the contents of each of which are expressly incorporated herein by reference. As another example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Genbank database. For example, the following heavy chain germline sequences found in HCo7 HuMAb mice are listed in the attached Genbank accession numbers 1-69 (NG--0010109, NT--024637 and BC070333), 3-33 (NG--0010109 and NT --024637) and 3-7 (NG--0010109 & NT--024637). As another example, the following heavy chain germline sequences found in HCo12 HuMAb mice are listed under attached Genbank accession numbers 1-69 (NG--0010109, NT--024637 and BC070333), 5-51 (NG-- 0010109 and NT--024637), 4-34 (NG--0010109 and NT--024637), 3-30.3 (CAJ556644) and 3-23 (AJ406678).

Antibody protein sequences were run against a compiled protein sequence database using one of the sequence similarity search methods known to those skilled in the art called Gapped BLAST (Altschul et al., (1997), supra). be compared.

Preferred framework sequences for use in the antibodies of the present disclosure are those that are structurally similar to the framework sequences used by the antibodies of the present disclosure. The V _H CDR1, CDR2, and CDR3 sequences can be grafted onto framework regions that have sequences identical to those found in the germline immunoglobulin genes from which the framework sequences are derived, or the CDR sequences can be grafted onto framework regions that have sequences identical to those found in the germline immunoglobulin genes from which the framework sequences are derived. may be grafted onto a framework region that contains one or more mutations compared to For example, in some cases it has been found beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of an antibody (e.g., U.S. Pat. No. 5,530,101 No. 5,585,089; No. 5,693,762 and No. 6,180,370).

Another type of variable region modification is to mutate amino acid residues within the V _H and/or V _L CDR1, CDR2 and/or CDR3 regions, thereby improving one or more binding properties of the antibody of interest. (e.g. affinity). Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations, and the effect on antibody binding, or other functional properties of interest, can be assessed in in vitro or in vivo assays known in the art. can be done. Preferably, conservative modifications (as known in the art) are introduced. Mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions. Additionally, typically no more than 1, 2, 3, 4 or 5 residues within a CDR region are modified.

Thus, in another embodiment, the present disclosure provides a V _H CDR1 region comprising (a) a sequence of the present disclosure, or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; b) a V _H CDR2 region comprising a sequence of the disclosure, or an amino acid sequence with 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions; (c) a sequence of the disclosure, or 1, 2, 3 , 4 or 5 amino acid substitutions, deletions or additions; (d) a sequence of the present _disclosure ; (e) a V _L CDR2 region comprising an amino acid sequence of the present disclosure, or having 1, 2, 3 _, 4 or 5 amino acid substitutions, deletions or additions; and (f ) An isolated anti-CTLA4 monoclonal comprising a heavy chain variable region comprising a V _L CDR3 region comprising a sequence of the present disclosure or an amino acid sequence having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions. Antibodies, or antigen-binding portions thereof, are provided.

Engineered antibodies of the present disclosure include those in which modifications have been made to framework residues within the V _H and/or V _L , eg, to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More particularly, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.

Another type of framework modification is to modify one or more within the framework region or within one or more CDR regions to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody. including mutating residues of. This technique, also referred to as "deimmunization", is described in further detail in US Patent Application Publication No. 2003/0153043.

In addition to, or in lieu of, modifications made within the framework or CDR regions, antibodies of the present disclosure typically improve serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cytotoxicity. Can be engineered to include modifications within the Fc region to alter one or more functional properties of the antibody, such as. Additionally, the antibodies of the present disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) to similarly alter one or more functional properties of the antibody or its glycosylation. can be modified to alter.

In one embodiment, the hinge region of C _H1 is modified such that the number of cysteine residues in the hinge region is altered, eg, increased or decreased. This approach is further described in US Pat. No. 5,677,425. The number of cysteine residues in the hinge region of C _H1 is varied, for example, to facilitate light and heavy chain assembly or to increase or decrease antibody stability.

In another embodiment, the Fc hinge region of the antibody is mutated to reduce the biological half-life of the antibody. More specifically, the one or more amino acid mutations alter the Fc-hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding compared to native Fc-hinge region SpA binding. It is introduced at the C _H2 -C _H3 domain boundary region. This approach is described in further detail in US Pat. No. 6,165,745.

In yet another embodiment, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be generated (ie, the antibody lacks glycosylation). Glycosylation can be modified, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such non-glycosylation can increase the affinity of the antibody for the antigen. See, eg, US Pat. No. 5,714,350 and US Pat. No. 6,350,861.

Additionally or alternatively, antibodies can be generated with altered types of glycosylation, such as hypofucosylated antibodies with a reduced amount of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase or decrease the ADCC capacity of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing antibodies in host cells with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies of the present disclosure, thereby producing antibodies with altered glycosylation. I can do it. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), and antibodies expressed in Ms704, Ms705, and Ms709 cell lines lack their It lacks fucose in carbohydrates. Ms704, Ms705, and Ms709 FUT8−/− cell lines were generated by targeted disruption of the FUT8 gene in CHO/DG44 cells using two replacement vectors (U.S. Patent Application Publication No. 2004/0110704 and Yamane et al. - See Ohnuki et al., (2004) Biotechnol Bioeng 87:614-22). As another example, European Patent No. 1,176,195 discloses that antibodies expressed in such cell lines have low fucosyl content by reducing or eliminating alpha-1,6 linkage-related enzymes. A cell line in which the FUT8 gene encoding fucosyltransferase has been functionally disrupted has been described. European Patent No. 1,176,195 also describes cells that bind to the Fc region of antibodies or that have low enzymatic activity for the addition of fucose to N-acetylglucosamine. Strains have been described, such as the rat myeloma cell line YB2/0 (ATCC CRL 1662). WO 03/035835 describes Lec13 cells, a mutant CHO cell line that has a reduced ability to bind fucose to Asn(297)-linked carbohydrates and also results in hypofucosylation of antibodies expressed in its host cells. (See also Shields et al., (2002) J. Biol. Chem. 277:26733-26740). Antibodies with modified glycosylation profiles can also be produced in chicken eggs, as described in WO 06/089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells such as Lemna. Methods for the production of antibodies in plant systems are disclosed in the corresponding US patent application filed Aug. 11, 2006, Alston & Bird LLP Attorney Docket No. 040989/314911. Fucose residues of antibodies can be cleaved using fucosidase enzymes; for example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al., (1975) Biochem. 14:5516-23 ).

Another modification of the antibodies herein contemplated by this disclosure is pegylation. Antibodies can be pegylated, for example, to increase the biological (eg, serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, is typically attached to a polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups are attached to the antibody or antibody fragment. (PEG). Preferably, pegylation is carried out by an acylation or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" refers to mono(C ₁ -C ₁₀ )alkoxy- or aryloxy-polyethylene glycols or polyethylene glycols used to derivatize other proteins such as maleimide. It is intended to include any of the forms of PEG that have been described. In certain embodiments, the antibody that is pegylated is a non-glycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See, for example, EP 154 316 and EP 0 401 384.

Antibodies of the present disclosure can be characterized by their various physical properties in order to detect and/or distinguish between their different classes.

For example, an antibody may contain one or more glycosylation sites in either the light chain or heavy chain variable region. Such glycosylation sites can result in increased immunogenicity of the antibody or modification of the pK of the antibody due to altered antigen binding (Marshall et al. (1972) Annu Rev Biochem 41:673-702; Gala and Morrison (2004) J Immunol 172:5489-94; Wallick et al. (1988) J Exp Med 168:1099-109; Spiro (2002) Glycobiology 12:43R-56R; Parekh et al. (1985) ) Nature 316:452- 7; Mimura et al., (2000) Mol Immunol 37:697-706). Glycosylation is known to occur in motifs containing NXS/T sequences. In some cases, it is preferable to have an anti-CTLA4 antibody that does not contain variable region glycosylation. This can be accomplished by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues within the glycosylation region.

In preferred embodiments, the antibody does not contain asparagine isomeric sites. Deamidation of asparagine can occur in the NG or DG sequence and can lead to the generation of isoaspartate residues that introduce bonds into the polypeptide chain and reduce its stability (isoaspartate effect). .

Each antibody has a unique isoelectric point (pI), generally within the pH range of 6-9.5. The pI of IgG1 antibodies is typically within the pH range of 7-9.5, and the pI of IgG4 antibodies is typically within the pH range of 6-8. It has been speculated that antibodies with pIs outside the normal range may have some unfolding and instability under in vivo conditions. Therefore, it is preferable to have an anti-CTLA4 antibody with a pI value within the normal range. This can be accomplished by selecting antibodies with pI within the normal range or by mutating charged surface residues.

In another aspect, the disclosure provides nucleic acid molecules encoding the heavy and/or light chain variable regions, or CDRs, of the antibodies of the disclosure. Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially pure" when it is purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. Nucleic acids of the present disclosure can be, for example, DNA or RNA, and may or may not include intronic sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

Nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For antibodies expressed by a hybridoma (e.g., a hybridoma prepared from a transgenic mouse carrying a human immunoglobulin gene described further below), the cDNA encoding the light and heavy chains of the antibody produced by the hybridoma is Obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (eg, using phage display technology), nucleic acids encoding such antibodies can be recovered from the gene library.

Preferred nucleic acid molecules of the present disclosure include those encoding the V _H and V _L sequences or CDRs of the CTLA4 monoclonal antibody. Once the DNA fragments encoding the V _H and V _L segments have been obtained, these DNA fragments can be subjected to standard assembly, e.g., to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. It can be further manipulated by engineered DNA techniques. In these manipulations, a DNA fragment encoding V _L - or V _H is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" when used in this context means that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame. It is intended that

The isolated DNA encoding the V _H region is prepared by operably linking the V _H encoding DNA to another DNA molecule encoding the heavy chain constant regions (C _H1 , C _H2 and C _H3 ). , can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but is most preferably an IgG1 or IgG4 constant region. For Fab fragment heavy chain genes, the DNA encoding the V _H can be operably linked to another DNA molecule encoding only the heavy chain C _H1 constant region.

The isolated DNA encoding the V _L region is generated by operably linking the V _L encoding DNA to another DNA molecule encoding the light chain constant region, C _L. and Fab light chain genes). The sequences of human light chain constant region genes are known in the art, and DNA fragments containing these regions can be obtained by standard PCR amplification. In preferred embodiments, the light chain constant region may be a kappa or lambda constant region.

To generate scFv genes, the V _H and V _L sequences encode V H - and _V L so that they can be expressed as a continuous single-chain protein with the V _L and V _H regions joined by a _flexible linker. A DNA fragment encoding a flexible linker is operably linked to another fragment encoding a flexible linker, eg, encoding the amino acid sequence (Gly4-Ser) 3 (eg, Bird et al., (1988) Science 242:423- 426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

Monoclonal antibodies (mAbs) of the present disclosure can be produced using the well-known somatic cell hybridization (hybridoma) technique of Kohler and Milstein (1975) Nature 256:495. Other embodiments for producing monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display technology. Chimeric or humanized antibodies are also well known in the art. For example, US Patent No. 4,816,567; US Patent No. 5,225,539; US Patent No. 5,530,101; US Patent No. 5,585,089; , 693,762 and 6,180,370, the contents of which are specifically incorporated herein by reference in their entirety.

Antibodies of the present disclosure can also be produced in host cell transfectomas using, for example, a combination of recombinant DNA technology and gene transfection methods as are well known in the art (e.g., Morrison, S. (1985) Science 229:1202). In one embodiment, DNA encoding partial or full-length light and heavy chains, obtained by standard molecular biology techniques, is isolated such that the genes are operably linked to transcriptional and translational regulatory sequences. It is inserted into the above expression vector. In this context, the term "operably linked" means that the antibody genes are ligated into the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating transcription and translation of the antibody genes. is intended to mean.

The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control the transcription or translation of antibody genes. Such regulatory sequences are described, for example, by Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression are promoters derived from viral elements, such as cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus, which direct high level protein expression in mammalian cells. and/or enhancers, such as the adenovirus major late promoter (AdMLP) and polyoma. Alternatively, non-viral regulatory sequences such as the ubiquitin promoter or the β-globin promoter may be used. Furthermore, regulatory elements are composed of sequences derived from different sources such as the SRα promoter system, including sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., (1988) Mol. Cell. Biol. 8:466-472). Expression vectors and expression control sequences are selected to be compatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene can be inserted into the same or separate expression vectors. In a preferred embodiment, the variable region has the desired shape, such that the V _H segment is operably linked to a C _H segment within the vector, and the V _L segment is operably linked to a C _L segment within the vector. It is used to generate full-length antibody genes for any antibody isotype by inserting the variable region into an expression vector that already encodes the heavy and light chain constant regions of the isotype. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide derived from a non-immunoglobulin protein).

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the present disclosure can have additional sequences, such as sequences that control replication of the vector in a host cell (eg, an origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (e.g., U.S. Pat. No. 4,399,216; U.S. Pat. No. 4,634,665 and U.S. Pat. No. 179,017). For example, typically the selectable marker gene confers resistance to drugs such as G418, hygromycin or methotrexate in a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

For expression of light and heavy chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. Various forms of the term "transfection" refer to a wide variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE - dextran transfection and the like. Although it is theoretically possible to express the antibodies of the present disclosure in either prokaryotic or eukaryotic host cells, eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to contain properly folded immunological molecules. Expression of the antibody in such eukaryotic cells, most preferably mammalian host cells, is most preferred because of the increased likelihood of assembling and secreting antibodies that are active in the host cell.

Preferred mammalian host cells for expressing recombinant antibodies of the present disclosure are Chinese hamster ovary (CHO cells) (e.g., R. J. Kaufman and P. A. Sharp (1982) J. Mol. Biol. 159: dhfr-CHO, as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, for use with the DHFR selectable marker, as described in NSO myeloma cells, COS cells and SP2 cells. In particular, for use with NSO myeloma cells, another preferred expression system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. It is a system. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody can be produced by expression of the antibody in the host cell or, more preferably, by secretion of the antibody into the culture medium in which the host cell is grown. produced by culturing host cells for a period sufficient to allow Antibodies can be recovered from the culture medium using standard protein purification methods.

The antibodies of the present disclosure, or antigen-binding portions thereof, can be conjugated to therapeutic agents to form immunoconjugates, such as antibody-drug conjugates (ADCs). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binders, DNA intercalating agents, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors. agents, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In an ADC, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys. , Lys, Cit, Ser, or a peptidyl linker such as Glu. ADC is disclosed in U.S. Patent No. 7,087,600; U.S. Patent No. 6,989,452; WO 07/051,081; WO 07/059,404; WO 08/083,312; and WO 08/103,693; US patent application As described in Publication No. 2006/0024317; Publication No. 2006/0004081; and Publication No. 2006/0247295 (the disclosures of which are incorporated herein by reference). can be prepared.

In another aspect, the present disclosure provides at least one other functional molecule, e.g., another peptide or protein (e.g., bispecific molecules comprising one or more antibodies of the present disclosure linked to another antibody or a ligand for a receptor). Thus, as used herein, "bispecific molecule" includes molecules with three or more specificities.

Bispecific molecules can be in many different formats and sizes. On one side of the size spectrum, bispecific molecules are similar to traditional antibody formats, except that instead of having two binding arms of the same specificity, they have two binding arms, each with a different specificity. is held. On the other side is a bispecific molecule consisting of two single-chain antibody fragments (scFv's) linked by a peptide chain, the so-called Bs(scFv) ₂ construct. Intermediate-sized bispecific molecules contain two different F(ab) fragments connected by a peptidyl linker. These and other formats of bispecific molecules can be prepared by genetic recombination, somatic cell hybridization, or chemical methods. For example, Kufer et al. (cited above); Cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al. , Immunology Today, 21(8), 391-397 (2000), and references cited therein.

Also provided herein are oncolytic viruses that preferentially infect and kill cancer cells. Antibodies of the present disclosure can be used with oncolytic viruses. Alternatively, oncolytic viruses encoding antibodies of the present disclosure can be introduced into the human body.

Also provided herein are chimeric antigen receptors (CARs) comprising anti-CTLA4 scFvs that include the CDRs and heavy chain/light chain variable regions described herein.

An anti-CTLA4 CAR can include (a) an extracellular antigen binding domain comprising an anti-CTLA4 scFv; (b) a transmembrane domain; and (c) an intracellular signaling domain.

CAR contains a signal peptide at the N-terminus of the extracellular antigen-binding domain that directs the nascent receptor to the endoplasmic reticulum, and a hinge at the N-terminus of the extracellular antigen-binding domain that makes the receptor more available for binding. May contain peptides. The CAR preferably includes a primary intracellular signaling domain and one or more co-stimulatory signaling domains. The primary and most effective major intracellular signaling domain used is the ITAM-containing CD3-ζ cytoplasmic domain, the phosphorylation of which results in T cell activation. Co-stimulatory signaling domains can be derived from co-stimulatory proteins such as CD28, CD137 and OX40.

CARs may further include factors that promote T cell proliferation, persistence, and anti-tumor activity, such as cytokines and co-stimulatory ligands.

Also provided are engineered immune effector cells comprising a CAR provided herein. In certain embodiments, the immune effector cells are T cells, NK cells, peripheral blood mononuclear cells (PBMCs), hematopoietic stem cells, pluripotent stem cells, or embryonic stem cells. In certain embodiments, the immune effector cells are T cells.

In another aspect, the present disclosure provides an antibody or antigen-binding portion thereof, bispecific antibody, CAR-T cell, oncolytic virus, immunoconjugate, nucleic acid molecule, expression vector, or host cell of the present disclosure. Provided is a pharmaceutical composition which can contain the above formulated together with a pharmaceutically acceptable carrier. When the composition contains two or more antibodies (or antigen-binding portions thereof, bispecific antibodies, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors or host cells), antibodies or antigen-binding portions thereof, bispecific antibodies, CAR-T cells, oncolytic viruses, immunoconjugates, nucleic acid molecules, expression vectors, or host cells can be administered separately. The composition may optionally contain one or more additional pharmaceutically active ingredients, for example agents such as other antibodies or anti-tumor agents.

Pharmaceutical compositions may include any excipients. Excipients that can be used include carriers, surfactants, thickeners or emulsifiers, solid binders, dispersion or suspension aids, solubilizers, colorants, flavorings, coatings, disintegrants, lubricants, Including sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients is described in Gennaro, ed. , Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Depending on the route of administration, the active ingredient may be coated with materials to protect it from the action of acids and other natural conditions that may render it inactive. As used herein, the phrase "parenteral administration" refers to methods of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, etc. Includes endo, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, intrathecal, intrathecal, epidural, and intrasternal injections and infusions. Alternatively, the antibodies of the present disclosure may be administered by a non-parental route, eg, by a topical, epidermal or mucosal route of administration, eg, intranasally, orally, vaginally, rectally, sublingually or topically.

Pharmaceutical compositions may be in the form of sterile aqueous solutions or dispersions. They can also be formulated in microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending on the subject treated and the particular mode of administration, and will generally be that amount of the composition that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.01% to about 99% of the active ingredient in combination with a pharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimal desired response (eg, therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subject being treated; each unit, together with the necessary pharmaceutical carrier, produces the desired therapeutic effect. Contains a predetermined amount of active ingredient calculated as follows. Alternatively, the antibody can be administered as a sustained release formulation if less frequent administration is required.

For administration of the composition, the dosage can range from about 0.0001 to 100 mg/kg host body weight.

A "therapeutically effective dose" of an anti-CTLA4 antibody, or antigen-binding portion thereof, or bispecific, CAR-T cell, oncolytic virus, immunoconjugate of the present disclosure preferably refers to the severity of disease symptoms. resulting in a reduction in the severity of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of disability or disability resulting from the affliction of the disease. For example, for the treatment of subjects with tumors, a "therapeutically effective dose" is preferably at least about 20%, more preferably at least about 40%, even more preferably at least about 60% compared to untreated subjects. %, even more preferably by at least about 80%. A therapeutically effective amount of a therapeutic antibody can reduce tumor size or ameliorate symptoms in a subject, which may typically be human or another mammal.

Pharmaceutical compositions can be controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. For example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed. , Marcel Dekker, Inc. , New York, 1978.

The therapeutic composition can be used in medical devices, such as (1) needleless subcutaneous injection devices (e.g., U.S. Pat. No. 5,399,163; U.S. Pat. No. 5,383,851; U.S. Pat. No. 5,312); , 335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556 ); (2) microinfusion pumps (U.S. Pat. No. 4,487,603); (3) transdermal devices (U.S. Pat. No. 4,486,194); (4) infusion devices (U.S. Pat. 4,447,233 and 4,447,224); and (5) osmotic devices (U.S. Pat. No. 4,439,196 and 4,475,196); (the disclosures of which are incorporated herein by reference).

In certain embodiments, monoclonal antibodies of the present disclosure can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic antibodies of the present disclosure cross the blood-brain barrier, they can be formulated in liposomes, which facilitate selective transport to specific cells or organs. It may further include a targeting molecule for targeting. For example, U.S. Pat. No. 4,522,811; U.S. Pat. No. 5,374,548; U.S. Pat. No. 5,416,016; V. Ranade (1989) J. Clin. Pharmacol. 29:685; Umezawa et al. , (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman et al. , (1995) FEBS Lett. 357:140;M. Owais et al. , (1995) Antimicrob. Agents Chemother. 39:180; Briscoe et al. , (1995) Am. J. Physiol. 1233:134; Schreier et al. , (1994) J. Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

The compositions of the present disclosure have numerous in vitro and in vivo utilities, including, for example, the treatment of cancer and infectious diseases. The compositions can be administered to human subjects, eg, in vivo, to inhibit tumor growth or to reduce or eliminate pathogens.

In another aspect, the present disclosure provides a method of treating or alleviating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of the present disclosure. Infectious diseases can be diseases caused by viral, bacterial, fungal or mycoplasma infections. In certain embodiments, the infectious disease is caused by chronic HIV or HHV-4 infection. In certain embodiments, the subject may further be administered at least one anti-infective agent, such as an anti-viral agent, an anti-bacterial agent, an anti-fungal agent, or an anti-mycoplasmal agent.

Given the ability of the anti-CTLA4 antibodies or antigen-binding portions of the present disclosure to reverse CTLA4-CD80/CD86-mediated T cell suppression and promote T cell responses, the present disclosure provides a method for inhibiting tumor growth in a subject. provided herein is a method of inhibiting tumor cell growth in a subject comprising administering to the subject a composition of the present disclosure. Non-limiting examples of tumors treatable by compositions of the present disclosure include, but are not limited to, melanoma, colorectal cancer, hepatocellular carcinoma, pleural mesothelioma, lung cancer (e.g., non-small cell lung cancer). ), renal cell carcinoma, cervical cancer, angiosarcoma, and muscle-invasive bladder cancer. Additionally, the antibodies of the present disclosure may be used to suppress the growth of refractory or recurrent malignant lesions.

In another aspect, the present disclosure provides an anti-CTLA4 antibody or antigen-binding portion thereof or bispecific antibody of the present disclosure, CAR-T, in combination with one or more additional antibodies effective to inhibit tumor growth in a subject. Methods of combination therapy are provided in which cells, oncolytic viruses, and immunoconjugates are co-administered. In one embodiment, the present disclosure provides anti-CTLA4 antibodies (or antigen-binding portions thereof or CAR-T cells, oncolytic viruses, immunoconjugates) as well as one or more additional antibodies, e.g., anti-VISTA antibodies, anti-LAG -3 antibody, an anti-PD-L1 antibody and/or an anti-PD-1 antibody is provided. In certain embodiments, the subject is a human.

CTLA4 signaling blockade can also be further combined with standard cancer treatments. For example, CTLA4 signaling blockade can be combined with LAG-3 and/or PD-1 blockade as well as chemotherapy regimes. For example, a chemotherapeutic agent can be administered with an anti-CTLA4 antibody that can be a cytotoxic agent. For example, epirubicin, oxaliplatin and 5-FU are administered to patients receiving anti-CTLA4 therapy.

Optionally, the combination of an anti-CTLA4 antibody and one or more additional antibodies (e.g., anti-TIM-3 and/or anti-LAG-3 and/or anti-PD-1 antibodies) is a combination of immunogenic agents, e.g. The cells can be further combined with cells transfected with purified tumor antigens (including recombinant proteins, peptides and carbohydrate molecules) and genes encoding immunostimulatory cytokines (He et al., (2004) J. Immunol .173:4919-28). Non-limiting examples of tumor vaccines that can be used include those transfected to express melanoma antigen peptides, such as gp100, MAGE antigen, Trp-2, MART1 and/or tyrosinase peptides, or the cytokine GM-CSF. These include tumor cells that have been treated with cancer.

Other therapies that may be combined with anti-CTLA4 therapy include, but are not limited to, interleukin-2 (IL-2) administration, radiation, surgery, or hormone deprivation.

In another aspect, the present disclosure provides methods of combination therapy in which an anti-CTLA4 antibody or antigen-binding portion thereof of the present disclosure is co-administered with one or more additional agents effective to reduce or eliminate a pathogen. provide. In one embodiment, the present disclosure is directed to an anti-CTLA4 antibody or antigen-binding portion thereof, as well as one or more additional agents against pathogens, such as antiviral, antibacterial, antifungal, or antimycoplasmal agents. A method of treating or ameliorating an infectious disease in a subject is provided, the method comprising: administering to a subject. In certain embodiments, the subject is a human.

The combinations of therapeutic agents described herein can be combined simultaneously as a single composition in a pharmaceutically acceptable carrier or as separate compositions containing each agent in a pharmaceutically acceptable carrier. can be administered. In another embodiment, the combination of therapeutic agents may be administered sequentially.

Additionally, when two or more doses of a combination therapy are administered sequentially, the order of sequential administration can be reversed or kept in the same order at each point of administration; sequential administration is defined as simultaneous administration. or any combination thereof.

The present disclosure is further illustrated by the following examples, which should not be construed as further limitations. The contents of all figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Example 1 Generation of Mouse Anti-CTLA4 Monoclonal Antibodies Using Hybridoma Technology Immunization Mice were purified by E. Harlow, D.; Lane, Antibody: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.C. Y. , 1998. Recombinant human CTLA4 protein (Acro biosystems, Cat #CT4-H5255) with a human IgG1 Fc tag at the C-terminus was used as the immunogen. Human CTLA4-his protein (Acro biosystems, Cat #CT4-H5229) was used to determine antiserum titers and to screen hybridomas secreting antigen-specific antibodies. The immunization dose included 25 μg human CTLA4-Fc protein/mouse/injection for both primary and booster immunizations. To increase the immune response, complete and incomplete Freund's adjuvant (Sigma, St. Louis, Mo., USA) were used for the primary and booster immunizations, respectively. Briefly, an adjuvant-antigen mixture was first prepared by gently mixing the adjuvant in a vial using a vortex. The desired amount of adjuvant was transferred to an autoclaved 1.5 mL microcentrifuge tube. Antigens were prepared in PBS or saline with concentrations ranging from 0.25 to 0.5 mg/ml. The calculated amount of antigen was then added to the microcentrifuge tube along with the adjuvant, and the resulting mixture was mixed by gentle vortexing for 2 minutes to produce a water-in-oil emulsion. The adjuvant-antigen emulsion was then drawn into a suitable syringe for injection into the animal. A total of 25 μg of antigen was injected in a volume of 100-200 μl. Each animal was immunized and then boosted 4-5 times depending on antiserum titer. Animals with good titers were given a final boost by intraperitoneal injection before fusion.

Hybridoma Fusion and Screening Cells of the mouse myeloma cell line (SP2/0-Ag14, ATCC #CRL-1581) were cultured to reach log phase stage just before fusion. Spleen cells derived from immunized mice were purified using the method described in Kohler G, and Milstein C, "Continuous cultures of fused cells secreting antibodies of predefined specificity", Nature. , 256:495-497 (1975). and fused with myeloma cells. The fused "hybrid cells" were then distributed into 96-well plates in DMEM/20% FCS/HAT medium. Viable hybridoma colonies were observed microscopically 7-10 days after fusion. Two weeks later, supernatants from each well were subjected to indirect ELISA using recombinant human CTLA4-his protein. Positive hybridomas secreting antibodies bound to human CTLA4-his protein were then selected and transferred to 24-well plates. Hybridomas were further tested by flow cytometry (FACS) for activity in blocking human CTLA4-Fc protein binding to cell surface CD80/CD86. Hybridoma clones producing antibodies that showed high specific human CTLA4 binding activity and CTLA4-Daudi cell blocking activity were subcloned by limiting dilution to ensure the clonality of the cell line, and then the monoclonal antibodies were purified. Briefly, a Protein A Sepharose column (bestchrom (Shanghai) Biosciences, Cat #AA0273) was washed with 5-10 column volumes of PBS buffer. The cell supernatant of the hybridoma monoclone was passed through the column, and the column was then washed with PBS buffer until the protein absorbance reached baseline. The column was eluted with elution buffer (0.1 M Glycine-HCl, pH 2.7) and collected immediately into a 1.5 ml tube containing neutralization buffer (1 M Tris-HCl, pH 9.0). Fractions containing immunoglobulins were pooled and dialyzed overnight at 4°C in PBS. The in vitro functional activity of the purified monoclonal antibodies was subsequently characterized as follows.

Example 2 Binding Affinity Determination of Mouse Anti-CTLA4 Monoclonal Antibodies Using BIACORE Surface Plasmon Resonance Purified anti-CTLA4 mouse monoclonal antibodies (mAbs) produced in Example 1 by the Biacore T200 system (GE healthcare, Pittsburgh, PA, USA) ) was characterized for binding affinity and kinetics.

Briefly, goat anti-mouse IgG (GE healthcare, Cat#BR100838, A Mouse Antibody Capture Kit) was covalently linked to a CM5 chip (a carboxymethyl dextran coated chip from GE healthcare #BR100530) and a Protein G chip (GE healthcare, Cat #29-1793-15) was used for benchmark affinity determination. used. Unreacted areas on the biosensor surface were blocked with ethanolamine. Next, purified anti-CTLA4 antibodies of the present disclosure at a concentration of 10 μg/ml and CTLA4 Benchmark (Bristol-Myers Squibb Co, Cat# NDC: 0003-2327-11, also referred to as Yervoy® or BM) were added at 10 μL/min. flowed onto the chip at a flow rate. Next, serially dilute recombinant human CTLA4-his (Acro biosystems, Cat# CT4-H5229, starting from 80 nM in 2-fold serial dilutions) or cynomolgus macaque CTLA4 in HBS-EP ⁺ buffer (provided by Biacore). -his protein (Acro biosystems, Cat #CT4-C5227, in 2-fold serial dilutions starting from 80 nM) was flowed onto the chip at a flow rate of 30 μL/min. The rate of antigen-antibody association was followed for 2 minutes and the rate of dissociation was followed for 10 minutes. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using BIAcore evaluation software. K _D , K _a and K _d values were determined and summarized in Table 2 below.

All mouse antibodies of the present disclosure specifically bound human and cynomolgus CTLA4 with similar or higher binding affinity compared to the benchmark. Antibodies C1D1, C1G4, D1D5, D1H4, D1B8 and D1A7 showed the highest level of binding affinity for human CTLA4.

Example 3 CTLA4 Binding Activity of Mouse Anti-CTLA-4 Antibodies The binding activity of mouse anti-CTLA4 antibodies of the present disclosure towards CTLA4 was determined by capture ELISA.

Briefly, 96-well plates were coated with AffiniPure goat anti-mouse IgG F(ab') ₂ fragment specific antibody (Jackson Immuno Research, Cat #115-005-072) at 2 μg/ml in PBS (100 μl/well). ) and incubated overnight at 4°C. Wash the plate once with washing buffer (PBS + 0.05% v/v Tween-20, PBST) and then add 200 μl/well of blocking buffer (5% w/v non-fat milk) for 2 hours at 37°C. PBST). The plate was washed four times and 100 μl/well of serially diluted mouse anti-CTLA4 antibody of the present disclosure, benchmark or negative control hIgG (intravenous human immunoglobulin (pH 4), Hualan Biological Engineering Inc.) (starting at 10000 ng/ml) was added. (5-fold dilution in PBST containing 2.5% w/v non-fat milk) for 40 minutes at 37°C and then washed again four times. 100 μl/well of biotinylated human CTLA4-Fc protein (Acro biosystems, Cat #CT4-H5255, 26 ng/ml in PBST with 2.5% w/v non-fat milk) containing captured anti-CTLA4 antibody was added to the plate containing captured anti-CTLA4 antibody. was added for 40 minutes at 37°C, washed 4 times, and incubated with streptavidin-conjugated HRP (1:10000 dilution in PBST, Jackson Immuno Research, Cat #016-030-084, 100 μl/well) at 37°C. and incubated for 40 minutes. After the final wash, plates were incubated with 100 μl/well of substrate TMB (Innoreagents, Cat #TMB-S-002). Stop the reaction within 15 min at room temperature with 50 μl/well of 1M H ₂ SO ₄ and read the absorbance of each well in a microplate reader using dual wavelength mode at 450 nm for TMB and 630 nm as reference wavelength. Read and then OD(450-630) values were plotted against antibody concentration. Data were analyzed using Graphpad Prism software and _EC50 values were reported. The results are shown in Figures 1A and 1B.

It can be seen from FIGS. 1A-1B that all mouse anti-CTLA4 antibodies of the present disclosure, except D1B8, specifically bind human CTLA4 with high binding capacity.

Example 4 Benchmark blocking activity and CTLA4-CD80/86 blocking activity of mouse anti-CTLA-4 antibody 4.1 Benchmark blocking ELISA
Benchmark - The ability of the disclosed anti-CTLA4 antibodies to block human CTLA4 binding was determined in a competitive ELISA assay. Briefly, benchmarks were coated onto 96-well microplates (100 μl/well) at 1.0 μg/mL in PBS and incubated for 2 hours at 37°C. Plates were washed once with wash buffer, blocked with 200 μl of PBST containing 5% w/v nonfat milk, incubated for 2 hours at 37° C., and washed 4 times.

Anti-CTLA4 antibodies of the present disclosure or controls were added to biotinylated human CTLA4-Fc (Acro biosystems, Cat #CT4-H5255, PBST containing 2.5% w/v non-fat milk) in 3-fold serial dilutions starting at 133.33 nM. (65 ng/ml) and incubated for 40 minutes at room temperature, then the antibody/human CTLA4-Fc mixture was added to the benchmark coated plates (100 μl/well). After incubation for 40 minutes at 37°C, the plates were washed again four times with wash buffer. The plates were then added with 100 μl/well of streptavidin-conjugated HRP (1:10000 dilution in PBST, Jackson Immuno Research, Cat #016-030-084, 100 μl/well) and incubated at 37° C. for 40 minutes. Plates were given a final wash using wash buffer. Finally, add TMB, stop the reaction using 1M _{H SO} and read the absorbance of each well in a microplate reader using dual wavelength mode at 450 nm for TMB and 630 nm as reference wavelength. , then OD(450-630) values were plotted against antibody concentration. Data were analyzed using Graphpad Prism software and _IC50 values were reported.

4.2 Cell-based ligand blocking FACS
The anti-CTLA4 antibodies of the present disclosure block human CTLA4-Fc protein binding to cell surface CD80/CD86 using the cell line Daudi (ATCC® CCL-213), which expresses cell surface human CD80 and human CD86. Activity was evaluated by flow cytometry (FACS).

An anti-CTLA4 antibody of the present disclosure, benchmark or negative control hIgG (intravenous human immunoglobulin (pH 4), Hualan Biological Engineering Inc.) was added to human CTLA4-Fc solution (Acro biosystems) at 2-fold serial dilution starting at 33.33 nM. , Cat#CT4-H5255, 1 μg/mL in FACS buffer) and incubated for 30 minutes at room temperature. Daudi cells were harvested from cell culture flasks in log phase, washed twice, and resuspended in PBS containing 2% v/v fetal bovine serum (FACS buffer). Daudi cells (1×10 ⁵ cells/well) were incubated for 40 minutes at 4° C. in 96-well plates containing 100 μl/well of antibody/CTLA4-Fc mixture. Wash the plate twice with FACS buffer, then add 100 μl/well of R-phycoerythrin AffiniPure goat anti-human IgG, Fcγ fragment-specific antibody (1:1000 dilution in FACS buffer, Jackson Immunoresearch, Cat #109-115- 098) and incubated for 40 minutes at 4°C in the dark. Cells were washed twice and resuspended in FACS buffer. Fluorescence was measured using a Becton Dickinson FACS Canto II-HTS instrument. Data were analyzed using Graphpad Prism software and _IC50 values were reported.

The results are shown in Figures 2A-2B and 3A-3B.

It can be seen from Figures 2A-2B that most antibodies of the present disclosure are able to block human CTLA4-benchmark binding, suggesting that these antibodies of the present disclosure bind to the same or similar epitope as the benchmark. Ru. Antibodies D2A4, D1B8 and D1H4 were unable to block human CTLA4 binding to the benchmark, suggesting that D2A4, D1B8 and D1H4 may bind to different epitopes.

Figures 3A-3B showed that most antibodies of the present disclosure were capable of blocking CTLA4 binding to cell surface CD80/CD86 with similar or higher activity than the benchmark.

Example 5 Cell-based Functional Assay of Mouse Anti-CTLA-4 Antibodies Anti-CTLA4 antibodies of the present disclosure were tested for activity in promoting T cell responses.

Briefly, in 20 μL of RPMI 1640 medium (Gibco, Cat #11875-093) supplemented with 10% FBS (Gibco, Cat #10099-141) and 5 μg/mL PHA (Sigma, Cat #L1668-5M). 4×10 ⁴ GS-J1 cells (immortalized human T lymphocytes expressing CD28, GenScript, Cat #M00611) were plated in each well of a 384-well plate (Corning, Cat #3707). Human CTLA4-Fc (Acro biosystems, Cat #CT4-H5255) was diluted to 8 μg/mL in RPMI 1640 medium supplemented with 10% FBS, and 20 μL of CTLA4-Fc was added to each well of the plate. Next, 2 × 10 ⁴ GS-C1/CD80 cells (immortalized antigen presenting cells expressing cell surface CD80, GenScript, Cat #M00614) in 20 μL of RPMI 1640 medium supplemented with 10% FBS, followed by 10% 20 μL of serially diluted anti-CTLA4 antibody (in 2.5-fold serial dilutions starting from 333.33 nM) in RPMI 1640 medium supplemented with FBS was added to each well of the plate. Plates were placed in a 5% CO ₂ incubator for 24 h at 37°C. Plates were centrifuged and IL2 levels in the supernatant were measured in 384-well small volume microplates (Greiner, Cat #784075) using the Human IL-2 HTRF kit (Cisbio, Cat #62HIL02PEG). Data were analyzed using Graphpad Prism software and _EC50 values were reported.

Figure 4 shows the assay results.

It can be seen that all antibodies of the present disclosure are capable of promoting T cell responses with slightly higher EC ₅₀ but similar maximum IL2 release levels compared to the benchmark.

Example 6 Generation and Characterization of Chimeric Antibodies The heavy and light chain variable domains of the anti-CTLA4 mouse mAb were sequenced and the SEQ ID numbers are summarized in Table 1.

The heavy and light chain variable domains of anti-CTLA4 mouse mAbs C1G4, D1B6, C1D1, D1D5 and D1B8 were combined with human IgG4 heavy chain constant region (SEQ ID NO: 78) and human kappa light chain constant region (SEQ ID NO: 79), respectively. They were cloned in frame and the C-terminus of the variable region was ligated to the N-terminus of each constant region.

A vector containing nucleotides encoding a heavy chain variable region linked to a human IgG4 heavy chain constant region (SEQ ID NO: 78) and a human kappa light chain constant region (SEQ ID NO: 79), respectively, using 1 mg/mL PEI. Vectors each containing nucleotides encoding linked light chain variable regions were transiently transfected into 200 ml of 293F suspension cell culture at a 1.1:1 heavy chain to light chain construct ratio.

After 6 days in shake flasks, the cell supernatant containing the chimeric antibody was harvested and the chimeric antibody was then purified from the cell supernatant as described above. Purified chimeric antibodies were tested in capture ELISA, Octet affinity testing, and cell-based ligand blocking FACS according to the protocols in the Examples above, with or without the modifications and protocols described below.

Purified anti-CTLA4 chimeric antibodies were characterized for binding affinity and kinetics by the Octet system (Fortebio, Octet RED 96). Briefly, the AHC biosensor (anti-human IgG Fc capture from ForteBio) was pre-soaked with 10 mM glycine (pH 1.5) for 3 seconds and then infused with running buffer (0.5% w/v in PBST). BSA) for 3 seconds, and the soaking and soaking process was repeated three times. The sensor was then immersed for 100 seconds in wells containing chimeric anti-CTLA4 antibody in 5 μg/ml HBS-EP ⁺ or benchmark in 5 μg/ml HBS-EP ⁺ and 5 min in wells containing running buffer. completely submerged. The new baseline was run for 180 seconds in a separate well containing running buffer. The sensor was then immersed for 100 seconds into a well containing serially diluted human CTLA4-his protein (Acro biosystems, Cat #CT4-H5229, starting at 80 nM in 2-fold serial dilutions) in running buffer; immersed in the baseline well for 10 min. Finally, the sensor was presoaked with 10 mM glycine (pH 1.5) for 3 seconds, then immersed in the well containing running buffer for 3 seconds, and the soak and dip steps were repeated three times. Association and dissociation curves were fitted to a 1:1 Langmuir binding model using ForteBio Data Analysis 8.1. K _a , K _d and K _D values were determined and summarized in Table 3 below.

In the capture ELISA, AffiniPure goat anti-human IgG, Fcγ fragment-specific antibody (Jackson Immuno Research, Cat #109-005-008) was used instead of AffiniPure goat anti-mouse IgG, F(ab') ₂ fragment-specific antibody. (100 μl/well).

The results are shown in Table 3 and FIGS. 5A to 5E and 6A to 6B.

As shown in Table 3, the chimeric C1G4, C1D1 and D1D5 anti-CTLA4 antibodies specifically bound human CTLA4 with higher binding affinity than the benchmark.

As shown in Figures 5A-5E and 6A-6B, the chimeric anti-CTLA4 antibodies had binding capacity and ligand blocking activity similar to their parental mouse mAbs. Chimeric D1B6, C1D1 and D1D5 anti-CTLA4 antibodies had slightly better blocking activity than Yervoy® in cell-based ligand blocking FACS.

Example 7 Humanization of anti-CTLA4 mouse monoclonal antibodies C1D1 and D1D5 Mouse anti-CTLA4 antibodies C1D1 and D1D5 were selected for humanization and further studies. Humanization of mouse antibodies was performed using well-established CDR grafting methods, as described in detail below.

To select receptor frameworks for humanization of murine antibodies C1D1 and D1D5, we blast searched the light chain and heavy chain variable region sequences of each murine antibody against the human immunoglobulin gene database. The human germline with the highest homology was selected as the receptor framework for humanization. Mouse antibody heavy/light chain variable region CDRs were inserted into the selected framework and residues within the framework were further backmutated to obtain more candidate heavy/light chain variable regions. A total of 12 humanized C1D1 antibodies, ie, huC1D1-V1 to huC1D1-V12, and 12 humanized D1D5 antibodies, ie, huD1D5-V1 to huD1D5-V12, were obtained. The heavy chain/light chain variable region sequence ID numbers are listed in Table 1.

A vector containing nucleotides encoding a humanized heavy chain variable region linked to a human IgG4 heavy chain constant region (SEQ ID NO: 78) and a human kappa light chain constant region (SEQ ID NO: 79), respectively, were isolated using 1 mg/mL PEI. ) were transiently translocated into 200 ml of 293F suspension cell culture at a 1.1:1 heavy chain to light chain construct ratio. transfected.

Example 8 Characterization of Humanized Antibodies After 6 days in shake flasks, cell supernatants containing humanized antibodies were harvested and incubated as described above with chimeric antibodies and benchmarks at a concentration of 5 μg/ml in HBS-EP ⁺ . Binding affinity to human CTLA4 was tested by Octet according to the protocol. K _a , K _d and K _D values were determined and summarized in Tables 4 and 5 below.

All cell supernatants containing humanized C1D1 antibodies showed higher binding affinity than the benchmark for human CTLA4, and cell supernatants containing huCTLA4 D1D5-V1 to huCTLA4 D1D5-V3 showed higher binding affinity for human CTLA4. The data suggested that the binding affinity was higher than that of the benchmark.

Humanized antibodies huC1D1-V8 and huD1D5-V9 were purified as described above and assayed for Biacore, capture ELISA, benchmark blocking ELISA, cell-based ligand blocking FACS and cell-based It was tested in a T cell response promotion test. In the capture ELISA assay, the goat anti-mouse IgG F(ab') ₂ fragment was replaced with 2 μg/ml goat anti-human IgG (AffiniPure goat anti-human IgG, F(ab') ₂ fragment-specific antibody, Jackson Immunoresearch, Cat #109-005-097) was coated in a 96-well microplate (100 μl/well). For the Biacore test, goat anti-human IgG (GE healthcare, Cat #BR100839, Human Antibody Capture Kit) was covalently linked to the CM5 chip instead of goat anti-mouse IgG, and for the benchmark CM5 instead of protein G chip. I used a chip.

The purified antibodies were also tested in a thermal stability assay using the GloMelt™ Thermal Shift Protein Stability Kit (Biotium, Cat #33022-T) to determine the Tm (melting temperature). Briefly, the GloMelt™ dye was thawed and allowed to reach room temperature. The vial containing the dye was vortexed and centrifuged. Next, 10X dye was prepared by adding 5 μL of 200X dye to 95 μL of PBS. 2 μL of 10× dye and 10 μg of humanized antibody were added, and PBS was added for a total reaction volume of 20 μL. The tubes containing dye and antibody were spun briefly and placed in a real-time PCR thermocycler (Roche, LightCycler 480 II) configured with a melting curve program with the parameters in Table 6.

The results are shown in Table 7 and FIGS. 7A and 7B, FIGS. 8A and 8B, FIGS. 9A and 9B, FIG. 10, and FIGS. 11A and 11B.

It can be seen from Table 7 that antibody huC1D1-V8 showed lower binding affinity for human CTLA4 and cynomolgus CTLA4 than the parent antibody or benchmark. This antibody effectively blocked CTLA4 binding to cell surface CD80/CD86, and the blocking activity was comparable when compared to the benchmark, as shown in Figure 9A.

Table 7 also shows that antibody huD1D5-V9 had equivalent binding affinity for human CTLA4 and cynomolgus monkey CTLA4. This antibody was shown in Figure 9B to effectively block CTLA4 binding to cell surface CD80/CD86 with slightly higher blocking potency when compared to the benchmark.

According to FIGS. 8A to 8B, the humanized antibodies huC1D1-V8 and huD1D5-V9 of the present disclosure were able to block human CTLA4-BM binding, so the antibodies huC1D1-V8 and huD1D5-V9 of the present disclosure It is suggested that it may bind to a similar epitope as BM.

As shown in Figure 10, humanized antibodies huC1D1-V8 and huD1D5-V9 of the present disclosure had comparable activity in promoting T cell responses when compared to BM.

Although the disclosure has been described above in conjunction with one or more embodiments, it should be understood that the disclosure is not limited to those embodiments, and the description is intended to be within the spirit and scope of the appended claims. It is intended to include all alternatives, modifications, and equivalents as may be included herein. All references cited herein are further incorporated by reference in their entirety.

The sequences in this application are summarized below.

While the preferred embodiments of the invention have thus been described in detail, the invention as defined by the foregoing paragraphs is capable of many obvious modifications thereof without departing from the spirit or scope of the invention. Therefore, it should be understood that there is no limitation to the specific details set forth in the above description.

Claims (15)

i) a heavy chain variable region comprising a VH CDR1 region, a VH CDR2 region and a VH CDR3 region, and
ii) a light chain variable region comprising a VL CDR1 region, a VL CDR2 region and a VL CDR3 region;
The amino acid sequences of the VH CDR1 region, the VH CDR2 region, the VH CDR3 region, the VL CDR1 region, the VL CDR2 region, and the VL CDR3 region are SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively . can be ,
An isolated monoclonal antibody or antigen-binding portion thereof capable of binding cytotoxic T lymphocyte-associated antigen 4 (CTLA4). The heavy chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of SEQ ID NO: 54 or 55. comprising amino acid sequences having % identity;
The 49th and 81st amino acid residues of SEQ ID NO: 55 are Ala (A) and Val (V), respectively; Ala (A) and Ala (A), respectively; Gly (G) and Val (V), respectively; or Gly (G) and Ala (A), respectively.
2. The isolated monoclonal antibody or antigen-binding portion thereof according to claim 1. The light chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% relative to SEQ ID NO: 56 or 57. comprising amino acid sequences having % identity;
The 43rd, 58th, 69th and 71st amino acid residues of SEQ ID NO: 57 are Ser (S), Met (M), Arg (R) and Tyr (Y), respectively; (V), Thr (T) and Phe (F); or respectively Val (V), Val (V), Thr (T) and Phe (F);
3. The isolated monoclonal antibody or antigen-binding portion thereof according to claim 1 or 2. The heavy chain variable region and the light chain variable region are
(1) Each of SEQ ID NOs: 54 and 56;
(2) In SEQ ID NOs: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Ala (A) and Val (V), respectively, and the 43rd amino acid residue of SEQ ID NO: 57 is The amino acid residues at positions 58, 69, and 71 are Ser (S), Met (M), Arg (R), and Tyr (Y), respectively ;
(3) In SEQ ID NO: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Ala (A) and Ala (A), respectively, and the 43 of SEQ ID NO: 57 is The amino acid residues at positions 58, 69, and 71 are Ser (S), Met (M), Arg (R), and Tyr (Y), respectively ;
(4) In SEQ ID NOs: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Gly (G) and Val (V), respectively, and the 43rd amino acid residue of SEQ ID NO: 57 is Gly (G) and Val (V), respectively. The amino acid residues at positions 58, 69, and 71 are Ser (S), Met (M), Arg (R), and Tyr (Y), respectively ;
(5) In SEQ ID NO: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Gly (G) and Ala (A), respectively, and 43 of SEQ ID NO: 57 is The amino acid residues at positions 58, 69, and 71 are Ser (S), Met (M), Arg (R), and Tyr (Y), respectively ;
(6) In SEQ ID NO: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Ala (A) and Val (V), respectively, and the 43rd amino acid residue of SEQ ID NO: 57 is The amino acid residues at positions 58, 69 and 71 are Ser (S), Val (V), Thr (T) and Phe (F), respectively ;
(7) In SEQ ID NO: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Ala (A) and Ala (A), respectively, and the 43 of SEQ ID NO: 57 is The amino acid residues at positions 58, 69 and 71 are Ser (S), Val (V), Thr (T) and Phe (F), respectively ;
(8) In SEQ ID NO: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Gly (G) and Val (V), respectively, and the 43rd amino acid residue of SEQ ID NO: 57 is Gly (G) and Val (V), respectively. The amino acid residues at positions 58, 69 and 71 are Ser (S), Val (V), Thr (T) and Phe (F), respectively ;
(9) In SEQ ID NO: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Gly (G) and Ala (A), respectively, and the 43rd amino acid residue of SEQ ID NO: 57 is Gly (G) and Ala (A), respectively. The amino acid residues at positions 58, 69 and 71 are Ser (S), Val (V), Thr (T) and Phe (F), respectively ;
(10) In SEQ ID NO: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Ala (A) and Val (V), respectively, and the 43rd amino acid residue of SEQ ID NO: 57 is The amino acid residues at positions 58, 69 and 71 are Val (V), Val (V), Thr (T) and Phe (F), respectively ;
(11) In SEQ ID NO: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Ala (A) and Ala (A), respectively, and the 43 of SEQ ID NO: 57 is The amino acid residues at positions 58, 69 and 71 are Val (V), Val (V), Thr (T) and Phe (F), respectively ;
(12) In SEQ ID NOs: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Gly (G) and Val (V), respectively, and the 43rd amino acid residue of SEQ ID NO: 57 is Gly (G) and Val (V), respectively. The amino acid residues at positions 58, 69 and 71 are Val (V), Val (V), Thr (T) and Phe (F), respectively ; or
(13) In SEQ ID NO: 55 and 57 , the 49th and 81st amino acid residues of SEQ ID NO: 55 are Gly (G) and Ala (A), respectively, and the 43 of SEQ ID NO: 57 is The amino acid residues at positions 58, 69 and 71 are Val (V), Val (V), Thr (T) and Phe (F), respectively ;
Claim 1 comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to The isolated monoclonal antibody or antigen-binding portion thereof according to any one of items 1 to 3. Claim comprising a heavy chain constant region having the amino acid sequence of SEQ ID NO: 78, linked to the heavy chain variable region, and a light chain constant region having the amino acid sequence of SEQ ID NO: 79, linked to the light chain variable region. 5. The isolated monoclonal antibody according to any one of 1 to 4, or an antigen-binding portion thereof. An isolated monoclonal antibody, or an antigen-binding portion thereof, according to any one of claims 1 to 5, which is a murine, chimeric or humanized antibody. 5. An isolated monoclonal antibody, or antigen-binding portion thereof, according to any one of claims 1 to 4 , which is of the IgG1, IgG2 or IgG4 isotype. A nucleotide encoding an isolated monoclonal antibody according to any one of claims 1 to 7, or an antigen-binding portion thereof. An expression vector comprising the nucleotide according to claim 8. A host cell comprising the nucleotide according to claim 8 or the expression vector according to claim 9. an isolated monoclonal antibody or an antigen-binding portion thereof according to any one of claims 1 to 7, a nucleotide according to claim 8, an expression vector according to claim 9 or a host cell according to claim 10; A pharmaceutical composition comprising: a pharmaceutically acceptable carrier. 12. A pharmaceutical composition according to claim 11 for use in inhibiting tumor growth in a subject in need thereof. The tumor is melanoma, colorectal cancer, hepatocellular carcinoma, pleural mesothelioma, lung cancer, renal cell carcinoma, cervical cancer, angiosarcoma, malignant pleural mesothelioma, metastatic urothelial tract cancer, or ureteral cancer. , urethral cancer, urinary tract cancer, head and neck cancer, squamous cell carcinoma, urothelial cell carcinoma, esophageal cancer, gastric cancer, gastroesophageal (GE) junction cancer, esophagogastric junction adenocarcinoma, anal cancer, bile duct cancer , Differentiated germ cell tumor, endometrial cancer, fallopian tube cancer, germ cell tumor, myelodysplastic syndrome, neuroblastoma, non-Hodgkin lymphoma, osteosarcoma, ovarian cancer, peritoneal cancer, prostate cancer, salivary gland cancer, sarcoma, triple Pharmaceutical composition for use according to claim 12, which is negative breast cancer (TNBC) or muscle invasive bladder cancer. 12. A pharmaceutical composition according to claim 11 for use in treating or alleviating an infectious disease in a subject in need thereof. Pharmaceutical composition for use according to claim 14, wherein the infectious disease is caused by chronic HIV infection or HHV-4 infection.

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